摘要
目的对81例假肥大型肌营养不良症（Duchenne/Becker muscular dystrophy,DMD/BMD）患者进行基因突变分析，探讨中国河南人群DMD基因突变的特点。方法收集81例无亲缘关系DMD／BMD患者的临床资料，应用多重连接依赖式探针扩增技术（multiplex ligation-dependent probe amplification, MLPA）对患者DMD基因进行外显子缺失／重复突变分析；对于MLPA检测为单个外显子缺失者，PCR扩增相应的外显子并进行Sanger测序以排除假阳性；对MLPA检测为阴性者，应用第二代测序（next generation sequencing,NGS）技术对79个外显子及其剪切位点进行测序，并用Sanger测序对微小突变进行验证。结果在81例DMD／BMD患者中，98．8％（80／81）检测出DMD基因缺失、重复或微小突变。MLPA技术检测出67例（82．7％，67／81）患者为DMD基因外显子缺失或重复突变，且第45～54外显子为缺失热点，其中79．1％（53／67）的患者临床表型符合“阅读框规则”；NGS和Sanger测序检测出13例患者为DMD基因点突变，其中新突变6个，已知致病突变7个。新突变中1个为移码突变（C．4708-4709insTG），5个为无义突变（c．8812G＞T、c．2131A＞T、c．6035T＞A、c．3426C＞A、c．3055C＞T）。结论本研究结果丰富了DMD基因突变谱；MLPA、NGS和Sanger测序相结合的检测流程提高了DMD基因检测的敏感度和特异性，对于进一步的遗传咨询、产前诊断和基因治疗都有重要意义。
Objective To perform mutation analysis for 81 unrelated patients with Duchenne/Becker muscular dystrophy （DMD/BMD） from Henan Province. Methods Multiplex ligation-dependent probe amplification （MLPA） was used to detect potential deletion/duplications of the DMD gene. Those with single exon deletions were validated with PCR amplification and Sanger sequencing to rule out false positive results. Patients with negative MLPA results were further analyzed with next-generation sequencing （NGS）, and the result was validated by Sanger sequencing. Results DMD gene deletion/duplications were detected in 67 cases by MLPA, and exons 45-54 was the most frequently deleted. The phenotypes of 79.1 patients with a deletion or duplication has conformed to the reading frame rule. In addition, 13 mutations were detected by NGS and Sanger sequencing, which included 6 novel mutations including one frameshift mutation c. 4708-4709insTG and 5 nonsense mutations （c. 8812G〉T, c. 213lAS〉T、c. 6035T〉A, c. 3426 C〉A, and e. 3055C〉T）. Conclusion This results have enriched the DMD gene mutation database. Combined MLPA, NGS and Sanger sequencing can greatly enhance the sensibility and specificity of genetic testing for the DMD/BMD.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2016年第6期762-767,共6页
Chinese Journal of Medical Genetics
基金
郑州市普通科技攻关项目20130958
关键词
假肥大型肌营养不良
DMD基因
二代测序
阅读框规则
Duchenne/Becker muscular dystrophy l DMD gene
Next-generation sequencing
The reading frame rule
